JP2814177B2 - DNA fragment containing alkaline pullulanase gene, vector incorporating the DNA fragment, and microorganism having the vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA fragment containing an alkaline pullulanase gene derived from an alkalophilic Bacillus bacterium, a vector incorporating the DNA fragment, and a transformant microorganism transformed with the vector. About.

[0002]

2. Description of the Related Art Pullulanase is present in a pullulan molecule.
Cleaving only the α-1,6 glucosidic bond
An enzyme that produces maltotriose, Bender and W
allenfelds [Biochem. Z., 334,
79 (1961)], Aerobacteria Aeroge
Nes (Aerobacter aerogenes) Or
Were discovered for the first time. Then, Bacillus sp.B
acillus sp. ) [J. Jpn. Soc. St
arch Sci., 30, 200 (1983)]
Chilus acid pruticalus(Bacillus a
Cidopullyticus)[Agric. B
iol. Chem., 52, 2293 (1984)],
Bacillus stearothermophilus (Bacillus
 stearothermophilus) [Eur.
J. Appl. Microbiol. Biotechn
ol., 17, 24 (1983)], Streptococcus
Smithis (Streptococcus miti
s) [Biochem. J., 108, 33 (196
8)], Lactobacillus (Lactobacillu
s) [Starch Science, 28, 72 (1981)], Crost
Rhidium thermohydrosulfuricam (Clostri
dium thermohydrosulfuricu
m) [Appl. Environ. Microb., 4
9, 5 (1985);Biochem. J., 246,
193 (1987)], Thermus SP (Ther
mus sp. ) [J. Jpn. Soc. Starch
Sci., 34, 1 (1987)]
Mu Thermosulfurogenes (Clostridium
thermosulfurogenes) [Appl.
Microb. Biotechnol., 33, 511
(1990)] can produce pullulanase.
It has been reported.

[0003] Pullulanase is known to have hydrolytic activity not only on pullulan but also on α-1,6 glucosidic bonds in starch, glycogen, amylopectin and branched oligosaccharides produced by partial degradation thereof.
It is called "branching enzyme". This enzyme can produce high yields of maltooligosaccharides such as glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose from starch in combination with endo- and exo-amylases. Have also been found and have recently been noted in the starch manufacturing industry.

On the other hand, the inventor of the present invention made use of the above-mentioned properties of pullulanase and blended pullulanase with α-amylase in dishwashing detergents and clothing detergents, thereby greatly improving the detergency against starch stains. A patent application was filed (Japanese Patent Application Laid-Open No. 2-132193) at the headline.

[0005] However, most of the pullulanases conventionally found in nature are classified as so-called neutral or acid pullulanases, which exhibit the maximum and stable enzyme activity in a neutral or acidic region.
The presence of pullulanases that exhibit maximum activity in the alkaline region or have alkali resistance, so-called alkali pullulanases or alkali-resistant pullulanases, which is a prerequisite for dishwashing and clothing cleaning compositions, is extremely low. Here, the alkaline pullulanase refers to those having an optimum pH in the alkaline region, and the alkali-resistant pullulanase has the optimum pH in the neutral to acidic region. A substance having a sufficient activity and maintaining stability. Neutral refers to a pH range of 6 to 8, and alkaline refers to a higher pH range.

[0006] As a method of producing conventionally known alkaline pullulanase and alkali resistant pullulanase, a method for producing alkali pullulanase by culturing an alkalophilic Bacillus bacteria Horikoshi et al (Bacillus s
p. 202-1) [Japanese Patent Publication No. 53-27786] and the present inventors ( Bacillus sp. KSM-18).
76) Only reported in Japanese Patent Application Laid-Open No. 3-87176.

[0007]

The gene for alkaline pullulanase produced by an alkalophilic Bacillus bacterium has not yet been obtained, and it is necessary to take genetic engineering means to produce it in large quantities, and to use the enzyme by a protein engineering technique. Was difficult to improve.

[0008]

[Means to solve the problem] To improve the titer,
Examination of cultivation conditions and breeding by sudden shift are commonly performed as means for improving productivity. On the other hand, a breeding method by genetic recombination, which has been remarkably developed in recent years, is attracting attention as a new method having a possibility of directly processing the gene. In breeding by this method, it is essential to first isolate the alkaline pullulanase gene. The present inventors aim at breeding by this method, and first, to obtain a DNA fragment containing an alkaline pullulanase gene from a chromosome, a recombinant Escherichia ( Es
A strain of the genus cherichia ) was prepared, and a DNA fragment containing the alkaline pullulanase gene was isolated from the strain.
As a result of comparing this DNA fragment with other pullulanase genes isolated from Bacillus bacteria, it was found that the DNA fragment was a novel DNA fragment having a unique restriction enzyme map, and the present invention was completed.

[0009] That is, the present invention is Bacillus sp. (Ba
Cillus sp. ) KSM-AP1378 (FER
MBP-3048), a DNA fragment comprising an alkaline pullulanase coding region and having a restriction map shown in FIG.
It is intended to provide a vector containing an NA fragment and a recombinant microorganism having the vector.

In the present invention, the microorganism serving as a donor of the alkaline pullulanase gene is, for example, Bacillus sp.
P1378 (FERM BP-3048). This strain has been isolated by the inventors as a strain that produces alkaline pullulanase Y having a significant amount of α-amylase activity outside the cells from soil in Tochigi City, Tochigi Prefecture,
Deposited as Micro-Engineering Research Deposit No. 3048. The taxonomic novelty of the strain is described in detail in a patent application (JP-A-3-108482) by the present inventors.

Bacillus sp. KS
As a method for obtaining chromosomal DNA from M-AP1378, the method of Marmur [ J. Mol. Biol . ,
3,208 (1961)] and the method of Saito and Miura [ Biochim. Biophys. Acta , 7
2, 619 (1963)], and other similar methods can also be used.

By cutting the prepared chromosomal DNA with a restriction enzyme, a DNA fragment containing an alkaline pullulanase gene can be obtained. The type of restriction enzyme used is a restriction enzyme having no cleavage site in the alkaline pullulanase producing gene. Anything can be used. In addition, all the restriction enzymes can be used if reaction conditions that cause only partial cleavage are used. Thus, various restriction enzymes can be selected according to the conditions used.

On the other hand, as a host / vector system used for gene recombination, the host strain can express the alkaline pullulanase gene, and the vector can be replicated in the host bacterium, so that the integrated gene can be stably used. Anything that can be held can be used. For example, Escherichia coli (Escherich
ia coli ) K-12 strain as host and Bacillus subtilis ( Bacillus subti).
lis ) BM strains using Marburg strains as hosts. As a specific example of the host strain, in the EK strain, H
B101 strain, C600 strain, JM109 strain and the like, and BM strains include BD170 strain, MI112 strain and the like. In addition to the above, if a vector that can be cut only at a single site by a restriction enzyme that cuts chromosomal DNA is used, it is convenient for binding to a chromosomal DNA fragment. Specifically, when chromosomal DNA is cut with Pst I,
In the case of the EK system, vectors such as pBR322, pUC12, and pUC18, and in the case of the BM system, vectors such as pUB110 and pBD8. It should be noted that other vectors having no restriction site by the restriction enzyme used for cutting chromosomal DNA were also ligated by the homopolymer binding method [Nelso].
n, T. and Brutlag, D.C. , Method
s in Enzymol. , 68, 41 (198
0)] and the like. The above chromosomal DNA fragment and the vector DNA cut with a restriction enzyme are ligated to prepare a recombinant plasmid. Examples of the ligating method include a method using DNA ligase and a homopolymer ligating method. Examples of DNA ligases include E. coli DNA ligase and T4
Examples include phage DNA ligase.

[0014] The recombinant vector thus prepared
There is no particular limitation on the method of transformation of the host strain by
Is, for example, in the case of an EK host strain, the calcium chloride method
[Mandel, M .; and Higa, A .; ,J. M
ol. Biol., 53, 159 (1970)] and chloride
Rubidium method [Bolivar, F .; and Back
man, k. ,Methods in Enzymo
l,68, 253 (1979)], and BM host bacteria
In the case of strains, the competent cell method [Content
e, S. and Dabnau, D .; ,Mol. Ge
n. Genet.177, 459 (1979)]
Rotoplast method [Chang, S .; and Cohe
n, S. N. ,Mol. Gen. Genet., 16
8, 111 (1978)].

The method for selecting and isolating from the transformant a microorganism into which a desired alkaline pullulanase-producing gene or a donor chromosomal DNA fragment containing an alkaline pullulanase-producing gene has been introduced is not particularly limited. Preferred is a method in which primary selection is carried out using the expression of such a marker, and then secondary selection is performed using the alkaline pullulanase activity of the host as an index. Specifically, for example, EK-based pBR is used as a vector plasmid.
322, and the chromosomal DNA was
When the Pst I cleavage fragment is inserted, the ampicillin gene containing this cleavage site is inactivated, so that primary selection may be performed using tetracycline resistance, which has no Pst I cleavage site in the gene, as an index. Then, as a secondary selection, a colored pullulan [Kano, M .; and
Tomiura, E .; , Agric. Biol. Che
m. , 49.1529 (1985)], and cultured by a replica method. After the colony appears, a strain that degrades pullulan around the colony and forms a zona pellucida is used as a target transformant. You can choose.

The recombinant plasmid of the transformant thus obtained can be prepared by a conventional plasmid or phage DNA preparation method [Maniatis, T .; et al. , Molec
ultra Cloning , (1982)]. By analyzing the cleavage pattern of the obtained recombinant plasmid with various restriction enzymes by electrophoresis or the like, the obtained recombinant plasmid can be used as a vector plasmid. It can be confirmed that the DNA fragment containing the alkaline pullulanase gene has been bound.
As shown in FIG. 1, the alkaline pullulanase gene of the present invention is contained in a DNA fragment of about 6.3 Kb whose both ends are Pst I cleavage sites.

A preferred example of the recombinant vector containing the alkaline pullulanase gene of the present invention is a recombinant plasmid pBP-101 (FIG. 1). The above plasmid is the Pst of the vector plasmid pBR-322.
About 10.7 kb DNA fragment containing the alkaline pullulanase gene shown in FIG.
It is a recombinant plasmid of Kb. FIG. 2 shows a detailed restriction map of the obtained Pst I fragment of the chromosomal DNA.

Examples of recombinant microorganisms transformed with the recombinant plasmid include Escherichia coli H
B101 (pBP-101) strain. This strain is obtained by introducing the recombinant plasmid pBP-101 into Escherichia coli HB101, and is capable of producing alkaline pullulanase by culturing in a medium usually used for culturing Escherichia bacteria, such as LB medium. The optimum reaction pH of the produced enzyme in the pullulanase activity is about 9.0 (FIG. 3), and it is clear that the enzyme is alkaline pullulanase.

[0019]

Examples of the present invention will be described below.

Example 1 Bacillus sp. KSM-AP137, a kind of alkalophilic Bacillus bacterium producing alkaline pullulanase
In preparing chromosomal DNA from No. 8 (FERM BP-3048), this strain was aerobically grown in 200 ml of the following medium I at 200C for 30 hours at 30 ° C.

[Table 1] After collection, the method of Saito and Miura [ Biochi
m. Biophys. Acta , 72, 619 (196
3)], about 2 mg of purified DNA was obtained.

Example 2 10 μg of the chromosomal DNA obtained in Example 1 and 1.5 μg of the vector plasmid pBR322 (manufactured by Boehringer Mannheim) were separately treated with a restriction enzyme reaction solution (10 mM Tris-base buffer (pH 7.5), 5 mM MgCl 2). ₂ · 7H
₂ O, 100 mM NaCl, 1 mM mercaptoethanol), add 10 units of restriction enzyme Pst I (Boehringer Mannheim), add 2.5 units at 37 ° C.
A time reaction was performed. The plasmid pBR322 is
Furthermore, 5'-phosphate residue was excised by alkaline phosphatase treatment. After the reaction, the restriction enzyme was removed by phenol treatment, and ethanol precipitation was performed.
Precipitated NA and cleaved vector plasmid pBR32
2 with a ligase reaction solution (20 mM Tris-HCl buffer (pH
7.5), 10 mM MgCl ₂ .7H ₂ O, 10 mM dithiothreitol, 1 mM ATP).
Two units of T ₄ DNA ligase (Boehringer Mannheim) were added thereto, and the mixture was reacted at 16 ° C. for 16 hours to perform a binding reaction between the chromosomal DNA and the vector plasmid.

Example 3 Transformation of Escherichia coli with the recombinant plasmid prepared in Example 2 was carried out by the calcium chloride method [Mandel, M .; an
d Higa, A .; , J. et al. Mol. Biol. , 53,
159 (1970)]. As the host bacteria, Escherichia coli HB101 strain (F ^- hsdS
20 recA13 ara-14 proA2 la
cY1 galK2 rpsL20 xyl-5 me
tl-1supE44 ) was used. 20 μg of the transformed cell suspension was treated with tetracycline (manufactured by Sigma).
LB agar medium containing 1.0 / ml (1.0% tryptone (Difco), 0.5% yeast extract (Difco), 0.
5% NaCl, 1.5% agar (manufactured by Wako Pure Chemical Industries, Ltd.)] and cultured at 37 ° C. for 16 hours. Next, about 10,
After replicating 000 transformant colonies on an LB agar medium (with tetracycline added) and growing them, 0.1 g of colonies were grown.
2% pullulan, 0.8% red pullulan [Kano,
M. and Tomomiura, E .; , Agric. Bi
ol. Chem. , 49, 1529 (1985)], 1
The agar containing mg / ml lysozyme was overlaid, and
Allowed to react for hours. The strain having the target alkaline pullulanase gene forms a transparent halo around the colony on this plate by the colored pullulan simple determination method. Thus, one clone having alkaline pullulanase productivity was obtained.

Example 4 The transformed strain obtained in Example 3 was inoculated into an LB liquid medium (with tetracycline added), pre-cultured at 37 ° C. overnight, and then 500 ml of an M9CA medium [0.6% Na ₂
HPO ₄ , 0.3% KH ₂ PO ₄ , 0.05% NaCl,
The cells were transplanted into 0.1% NH ₄ Cl, 0.2% casamino acid (manufactured by Difco), 2 mM MgSO ₄ .7H ₂ O, 0.2% glucose, 20 μg / ml tetracycline, and 37 ° C.
For 4 to 5 hours with shaking. To this was added 170 mg of chloramphenicol, followed by shaking at 37 ° C. for 15 hours. The cells were collected from this culture by centrifugation and subjected to an alkaline lysis method [Birnboim, H .; C. and Do
ly, J.L. , Nucleic Acids Res. ,
7, 1513 (1979)] and cesium chloride-
Ethidium bromide density gradient centrifugation [Radlof
f, R. , Bauer, W .; and Vinogra
d. , Proc. Natl. Acad. Sci. U
SA, 57, 1514 (1967)] [Maniatis, T .; et al. , Molec
according to U.S.A. , US Pat .
A recombinant plasmid was prepared (FIG. 1). Further, in order to carry out detailed analysis, after various restriction enzymes were allowed to act on this plasmid, the cleavage pattern was analyzed by agarose gel electrophoresis. Based on the results, a restriction enzyme map (FIG. 2) was prepared. As a result, this plasmid contained a pullulanase gene known to date, for example, Bacillus stearothermophilus thermostable pullulanase (Japanese Unexamined Patent Publication No.
No. 23872) and the like, showing a different restriction map. Therefore, this plasmid was named pBP-101, and the Escherichia coli HB101 strain transformed thereby was named Escherichia coli HB101 (pBP-101).

Example 5 Escherichia coli HB101 (pBP-101) was cultured in an LB liquid medium (with tetracycline), and the collected cells were suspended in a Tris-HCl buffer (pH 8.0). Crushing was performed. Again, insolubles were removed as a precipitate by centrifugation, and the resulting supernatant was used as a cell-free extract. As a control, HB101 (pBR-322)
Cell-free extracts were similarly prepared for the strains, and their pullulanase activities were measured. Pullulanase activity was measured by adding 0.1 ml of the enzyme solution to 0.9 ml of a substrate solution in which 40 mM glycine-salt-sodium hydroxide buffer (pH 10) and pullulan (final concentration 0.25%) were dissolved,
The reaction was carried out for 30 minutes and 3,5-dinitrosalicylic acid (3,5
5-dinitro-salicyclic acid
(DNS)) method to quantify reducing sugars. That is, 1.0 ml of the DNS reagent was added to 1.0 ml of the reaction solution, and
After heating for 5 minutes, the mixture was cooled, diluted with 4.0 ml of deionized water, and colorimetrically determined at a wavelength of 535 nm. The enzyme titer is 1
The amount of the enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute was defined as one unit. As a result, as shown in Table 2, alkaline pullulanase activity was observed in the cell-free extract of the HB101 (pBP-101) strain.

[0025]

[Table 2]

Example 6 Using the cell-free extract obtained in Example 5, the optimum pH of the produced pullulanase was measured. The enzyme activity was measured using the buffers shown below (40 mM each), and
The measurement was performed in the same manner as described above. pH 3.5-5.5 acetate buffer pH 5.5-8.5 trismalate buffer pH 8.5-10.5 glycine-salt-sodium hydroxide buffer pH10.5-11.0 carbonate buffer As a result, the pullulanase activity of this enzyme was pH 9.0.
Was found to be an alkaline pullulanase having an optimum action pH (FIG. 3).

[0027]

As described above, the use of the DNA fragment of the present invention makes it possible to produce a large amount of alkali pullulanase useful as a component of a detergent at low cost.

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for constructing a recombinant plasmid pBP101 prepared using a vector plasmid pBR322. The portion corresponding to the 6.3 Kb Pst I fragment shown by the thick line in the figure is Bacillus sp. KSM-AP13.
This is a portion containing an alkaline pullulanase gene derived from 78 strains.

FIG. 2 is a diagram showing a restriction map of pBP101.

FIG. 3. Recombinant Escherichia coli HB101 (pB
It is a figure which shows the action pH of pullulanase activity which the P-101) strain produces, and the optimal action pH.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 9/44 C12R 1:19) (C12N 15/09 C12R 1:07) (56) References Biosci. Biotechnol l. Biochem. 56 (1) p. 62-65 (1992) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/56 C12N 1/21 C12N 9/44 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI / L (QUESTEL)

Claims (3)

(57) [Claims] [Claim 1] Bacillus sp. (Bacillus
sp. ) KSM-AP1378 (FERM BP-3
048), containing the alkaline pullulanase coding region isolated from 6.3) and having the restriction map shown in FIG.
A DNA fragment consisting of Kb base pairs. 2. A recombinant vector containing the DNA fragment according to claim 1. A microorganism having the recombinant vector according to claim 2.